The long term goal of our research is to understand the mechanisms of proton pumping ATPases. Neurospora crassa serves as a model system from which we can isolate three ATPases representative of the three types found in virtually all eucaryotic cells. The mitochondria contain a typical F0F1-ATPase. The plasma membrane contains an H+-ATPase very similar in structure and mechanism to the Na+,K+-ATPase, Ca+2-ATPase, and H+,K+-ATPase of animal cells. The vacuolar membrane contains a newly characterized type of H+-ATPase. On the basis of inhibitor sensitivity and kinetic data, the vacuolar enzyme closely resembles the H+-ATPases of animal cell lysosomes and secretory granules. The major emphasis in our proposed research is to understand the structure and function of the vacuolar ATPase. Our data indicate that the enzyme is a large multisubunit complex. We plan to purify the enzyme further and determine the size and number of copies of each of the subunits. We propose to examine the nature of the "proton channel" in the vacuolar ATPase. This enzyme contains an Mr = 15,000 subunit which binds dicyclohexylcarbodiimide (DCCD). We wish to identify the DCCD binding residue and determine if the polypeptide is a proteolipid. We also plan to measure the electrochemical gradient generated by the ATPase and to evaluate the nature of its coupling to amino acid uptake in vacuoles. We have isolated polyclonal antibodies to the two major subunits of the vacuolar ATPase. These, and monoclonal antibodies now being selected, will be used in the purification and structural analysis of the ATPase. A second major project with the antibodies is the cloning of the vacuolar ATPase genes, using antibodies to screen a cDNA library constructed in phage lambda gt11. From our initial experiments, we may already have the cDNA for one of the subunits. Much of our past work was devoted to purifying and characterizing the H+-ATPase in the plasma membrane, and we plan to pursue two aspects of that work. First, the hypothesis that the plasma membrane enzyme is a dimer with interacting subunits will be tested by crosslinking the enzyme with reagents that bind specific residues. Since the sequence of this enzyme is known, we can attempt to identify regions of the polypeptide involved in subunit interactions. Second, we have two new strategies to search for mutants with altered plasma membrane ATPase.